Determinants of increased replicative capacity of serially passaged simian immunodeficiency virus with nef deleted in rhesus monkeys

Abstract

Most rhesus macaques infected with simian immunodeficiency virus SIVmac239 with nef deleted (either Delta nef or Delta nef Delta vpr Delta US [Delta 3]) control viral replication and do not progress to AIDS. Some monkeys, however, develop moderate viral load set points and progress to AIDS. When simian immunodeficiency viruses (SIVs) recovered from two such animals (one Delta nef and the other Delta 3) were serially passaged in rhesus monkeys, the SIVs derived from both lineages were found to consistently induce moderate viral loads and disease progression. Analysis of viral sequences in the serially passaged derivatives revealed interesting changes in three regions: (i) an unusually high number of predicted amino acid changes (12 to 14) in the cytoplasmic domain of gp41, most of which were in regions that are usually conserved; these changes were observed in both lineages; (ii) an extreme shortening of nef sequences in the region of overlap with U3; these changes were observed in both lineages; and (iii) duplication of the NF-kappa B binding site in one lineage only. Neither the polymorphic gp41 changes alone nor the U3 deletion alone appeared to be responsible for increased replicative capacity because recombinant SIVmac239 Delta nef, engineered to contain either of these changes, induced moderate viral loads in only one of six monkeys. However, five of six monkeys infected with recombinant SIVmac239 Delta nef containing both TM and U3 changes did develop persisting moderate viral loads. These genetic changes did not increase lymphoid cell-activating properties in the monkey interleukin-2-dependent T-cell line 221, but the gp41 changes did increase the fusogenic activity of the SIV envelope two- to threefold. These results delineate sequence changes in SIV that can compensate for the loss of the nef gene to partially restore replicative and pathogenic potential in rhesus monkeys.

FIG. 1.

Lineage of rhesus monkeys experimentally infected with SIVΔnef. The arrows indicate the history of the SIV passage. The viral loads represent SIV RNA copies/ml of plasma. 5′ and 3′ SIV DNA refers to the PCR-generated fragments that were used to generate a viral stock as described in Materials and Methods. LPD, lymphoproliferative disease.

FIG. 2.

Lineage of rhesus monkeys experimentally infected with SIVΔ3. The arrows indicate the history of the SIV passage. The viral loads represent SIV RNA copies/ml of plasma. 5′ and 3′ SIV DNAs generated by PCR were used to generate viral stocks as described in Materials and Methods. RRV, rhesus monkey rhadinovirus.

FIG. 3.

Alignment of SIVmac239, cloned SIVmac recombinant, and monkey-passaged SIVmac sequences. The stars denote nucleotides or amino acids that were homologous with those of SIVmac239, and the dashes denote nucleotides or amino acids that were not contained in a particular sequence. (A) Amino acid alignment of gp41 cytoplasmic tail sequences of SIVmac239 and monkey-passaged sequences isolated from an animal from the Δnef lineage (Mm66-95) and an animal from the Δ3 lineage (Mm138-95). Mm8-22 represents the env sequence used in the lineage leading to Mm66-95. The number 721 denotes the amino acid position in Env in the SIVmac239 strain (57). Areas in grey denote sequences that were invariant in the Los Alamos database that were altered in Mm66-95 and Mm138-95. (B) Nucleotide alignment of nef sequences in the region of overlap with U3 (US). (C) Nucleotide alignment of LTR sequences in the region in which a duplication polymorphism was observed in SIV isolated from Mm138-95. NF-κB binding sites are shown in bold letters.

FIG. 4.

Quantification of SIV loads in rhesus monkeys experimentally infected with SIVΔnef. (A) Plasma SIV RNA levels at the indicated weeks postinoculation. The dashed line indicates the threshold sensitivity of the assay (100 copies/ml). (B) Frequency of infectious cells in PBMC. Viral loads were graded on a scale of 0 to 10, indicating the number of PBMC needed to recover SIV: 0, no virus recovered from 10⁶ cells; 1, successful virus recovery from 10⁶ cells; 2 to 10, successful virus recovery from 333,333, 111,111, 37,037, 12,345, 4,115, 1,371, 457, 152, and 51 cells, respectively.

FIG. 5.

Quantification of SIV loads in rhesus monkeys experimentally infected with SIVΔnefΔUS138. (A) Plasma SIV RNA levels at the indicated weeks postinoculation. The dashed line indicates the threshold sensitivity of the assay (1,500 copies/ml). (B) PBMC-associated viral load. Viral loads were graded on a scale of 0 to 10, indicating the number of PBMC needed to recover SIV as described in the legend to Fig. 4.

FIG. 6.

Quantification of SIV loads in rhesus monkeys experimentally infected with SIV/ITMΔnefΔUS138KK. (A) Plasma SIV RNA levels at the indicated weeks postinoculation from this animal experiment. The dashed line indicates the threshold sensitivity of the assay (1,500 copies/ml). (B) Same as in A for a separate animal experiment. The dashed line indicates the threshold sensitivity of the assay (100 copies/ml). (C) PBMC-associated viral loads. (D) PBMC-associated viral loads.

FIG. 7.

Quantification of SIV loads of rhesus monkeys experimentally infected with SIV/ITMΔnefΔUS138. (A) Plasma SIV RNA levels at the indicated weeks postinoculation. The dashed line indicates the threshold sensitivity of the assay (1,500 copies/ml). (B) PBMC-associated viral loads. Viral loads were graded on a scale of 0 to 10, indicating the number of PBMC needed to recover SIV.

FIG. 8.

Quantification of SIV loads of rhesus monkeys experimentally infected with SIV/ITMΔnefΔUS138KK. (A) Plasma SIV RNA levels at the indicated weeks postinoculation. (B) Same as in A but for a separate animal experiment. (C) PBMC-associated viral loads. (D) PBMC-associated viral loads.

FIG. 9.

Replicative capacity of SIV recombinants in unstimulated 221 cells. The cells were incubated without interleukin-2 and infected with virus diluted to contain 10 ng of p27^Gag antigen. p27^Gag antigen production from infected cultures was quantified at the times indicated.

FIG. 10.

Fusogenic activity of SIVmac239 and monkey-passaged gp41 transmembrane (TM) sequences. 293T cells were transfected with pLTR/CTM/GFP (containing SIVmac239 TM sequences) and pLTR/ITM/GFP (containing monkey-passaged TM sequences) and incubated with C8166-45 LTR-SEAP cells. At the times indicated, cells were analyzed for GFP expression and supernatants were harvested for measurement of secreted alkaline phosphatase (SEAP) activity. The data are expressed as relative light units (RLU) of SEAP or GFP. The data represent two independent quantifications, each from two separate transfection experiments.